Compositions and method of use of a-type procyanidins

ABSTRACT

The invention relates to compositions, such as pharmaceuticals, foods, food additives, or dietary supplements, containing A-type procyanidins, and methods of use thereof, for prophylactic or therapeutic treatment of a human or a veterinary animal to treat or prevent NO-responsive health conditions, treat hypertension, cardiovascular disease, coronary artery disease and/or vascular circulation disorders, prevent or reduce the risk of heart attack, stroke, congestive heart failure and/or kidney failure, or to improve blood flow, for example renal blood flow. The composition may optionally contain an additional NO modulating agent and/or a cardiovascular-protective or therapeutic agent, or may be administered in combination with such an agent.

This application claims the benefit, under 35 USC Section 119, of theU.S. Provisional Appl. No. 60/539,689 filed Jan. 28, 2004, thedisclosure of which is hereby incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to compositions containing A-type procyanidins,and methods of use thereof, for prophylactic or therapeutic treatment ofa human or a veterinary animal.

BACKGROUND OF THE INVENTION

Polyphenols are an incredibly diverse group of compounds (Ferriera etal., Tetrahedron, 48:10, 1743-1803, 1992). They widely occur in avariety of plants, some of which enter into the food chain. In somecases they represent an important class of compounds for the human diet.Although some of the polyphenols are considered to be non-nutritive,interest in these compounds has arisen because of their possiblebeneficial effects on health. For instance, quercetin (a flavonoid) hasbeen shown to possess anticarcinogenic activity in experimental animalstudies (Deshner et al., Carcinogenesis, 7:1193-1196, 1991: and Kato etal., Carcinogenesis, 4, 1301-1305 1983). (+)-catechin and(−)-epicatechin (flavan-3-ols) have been shown to inhibit Leukemia virusreverse transcriptase activity (Chu et al., J. of Natural Prod., 55:2,179-183, 1992). Nobotanin (an oligomeric hydrolysable tannin) has alsobeen shown to possess anti-tumor activity (Okuda et al., presented atthe XVIth International Conference of the Groupe Polyphenols, Lisbon,Portugal, Jul. 13-16, 1992). Procyanidin oligomers have been reported bythe Kikkoman Coporation for use as antimutagens (JP 04190774A, Jul. 7,1992).

Some polyphenol, such as B-type procyanidins have been shown to havebeneficial effect on nitric oxide (NO) release and hence on treatment ofa variety of health conditions that positively respond to NO (see e.g.U.S. Pat. No. 6,670,390 to Romanczyk et al.).

Nitric oxide (NO) is known to inhibit platelet aggregation, monocyteadhesion and chemotaxis, and proliferation of vascular smooth muscletissue which are critically involved in the process of atherogenesis.The concentration of NO can be reduced in atherosclerotic tissues due toits reaction with oxygen free radicals. The loss of NO due to thesereactions leads to increased platelet and inflammatory cell adhesion tovessel walls to further impair NO mechanism of relaxation. In thismanner, the loss of NO may promote atherogenic processes, leading toprogressive disease states.

Hypertension is a condition where the pressure of blood as it circulateswithin the blood vessels is higher than normal. When the systolicpressure exceeds 150 mm Hg or the diastolic pressure exceeds 90 mm Hgfor a sustained period of time, damage is done to the body. Hypertensionis a leading cause of vascular diseases, including stroke, heart attack,heart failure, and kidney failure. For example, excessive systolicpressure can rupture blood vessels anywhere. In cases when a raptureoccurs within the brain, a stroke results. Hypertension can also causethickening and narrowing of the blood vessels which can lead toatherosclerosis. Elevated blood pressure can also force the heat muscleto enlarge as it works harder to overcome the elevated resting(diastolic) pressure when blood is expelled. This enlargement caneventually produce irregular heart beats or heart failure. Hypertensionis called the “silent killer” because it causes no symptoms and can onlybe detected when blood pressure is checked.

The regulation of blood pressure is a complex event where one mechanisminvolves the expression of constitutive (Ca+2/calmodulin dependent formof nitric oxide synthase (NOS), known as endothelial nitric oxidesynthase or eNOS. NO produced by this enzyme produces smooth musclerelaxation in the vessel (dilation), which lowers the blood pressure.When circulating concentrations of NO are reduced, either becauseproduction is blocked by an inhibitor or in pathological states, such asatherosclerosis, the vascular muscles do not relax to the appropriatedegree. The resulting vasoconstriction increases blood pressure and maybe responsible for some forms of hypertension.

Given the large number of people suffering from hypertension and relateddiseases and disorders of the vascular systems, there is considerableinterest in finding therapeutic ways to maintain the NO pool at itsnormal, healthy levels. Pharmacological agents capable of releasing NO,such as nitroglycerin or isosorbide dinitrate, remain mainstays ofvasorelaxant therapy. Applicants have now surprisingly discovered thatA-type procyanidins can be utilized for preserving the NO pool, inducingvasorelaxation and/or treating and/or preventing NO-responsive diseasesand disorders.

SUMMARY OF THE INVENTION

The invention relates to compositions containing A-type procyanidins,and methods of use thereof, for prophylactic or therapeutic treatment ofa human or a veterinary animal.

In one aspect, the invention relates to a composition, such as apharmaceutical, a food, a food additive, or a dietary supplementcomprising an effective amount of an A-type procyanidin. The compositionmay optionally contain an additional NO modulating agent and/or acardiovascular-protective or therapeutic agent, or may be administeredin combination with such an agent. Also within the scope of theinvention are packaged products containing the above-mentionedcompositions and a label and/or instructions for use to treat or preventNO-responsive health conditions, treat hypertension, cardiovasculardisease, coronary artery disease and/or vascular circulation disorders,prevent or reduce the risk of heart attack, stroke, congestive heartfailure and/or kidney failure, or to improve blood flow, for examplerenal blood flow.

In another aspect the invention relates to methods of use of A-typeprocyanidins to treat or prevent NO-responsive health conditions, treathypertension, cardiovascular disease, coronary artery disease and/orvascular circulation disorders, prevent or reduce the risk of heartattack, stroke, congestive heart failure and/or kidney failure, or toimprove blood flow, for example renal blood flow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C represent dose-dependent relaxation mediated by A-typeprocyanidins in pre-contracted aortic rings: A2 dimer (1A); A1 dimer(1B); and A-type trimer (1C).

FIGS. 2A-2D represent the results of platelet aggregation experimentswith A1 dimer: aggregation at 4 minutes (2A) and 10 minutes (2B); P/M(2C) and P/N (2D) measured in response to all collagen concentrations(0.125, 0.25, and 0.5 μg/ml) in the absence and presence of a range offlavanols and ASA (100 μM). Results=mean±S.E.M. n=9 determinations.*p<0.05 cf control, $p<0.05 cf ASA.

FIGS. 3A-3G represent the results of platelet aggregation experimentswith A1 dimer: platelet aggregation (3A), P/M (3B), P/N (3C), CD62P-M(3D), CD62P-N (3E), CD11b-M (3F), and CD11b-N (3G) measured in responseto all collagen concentrations (0.125, 0.25 and 0.5 μg/ml) in theabsence and presence of a range of flavanols and ASA (100 μM).Results=mean±S.E.M. n=9 determinations. *p<0.05 cf control, $p<0.05 cfASA.

DETAILED DESCRIPTION

All patents, patent applications and references cited in thisapplication are hereby incorporated herein by reference. In case of anyinconsistency, the present disclosure governs.

The invention relates to compositions comprising an effective amount ofan A-type procyanidin, or a pharmaceutically acceptable salt orderivative thereof.

The A-type procyanidin of the present invention is an oligomer composedof n monomeric, flavan-3-ol units of the formula:

wherein

-   (i) the monomeric units are connected via interflavan linkages 46    and/or 4 8;-   (ii) at least two of the monomeric units are additionally linked by    an A-type interflavan linkage (4 8; 2 0 7) or (4 6; 2 0 7); and-   (iii) n is 2 to 12.

It will be understood by a person of skill in the art that one of thetwo flavanol units linked by the A-type interflavanoid linkage mustcomprise two bonds at the 2- and 4-positions. Both of these have eitherα or β stereochemistry, i.e., the bonds are either 2α, 4α or 2β, 4β.These bonds connect to the 6- and 7-O-positions, or the 8- and7-O-positions of the second flavanol unit linked by the A-typeinterflavan linkage. In constituent flavanol units of the oligomer whichdo not comprise A-type interflavan linkages at positions C-2 and C-4,the linkage at position C-4 can have either alpha or betastereochemistry. The OH group at position C-3 of flavanol units haseither alpha or beta stereochemistry. Flavan-3-ol (monomeric) units maybe (+)-catechin, (−)-epicatechin and their respective epimers (e.g.(−)-catechin and (+)-epicatechin)).

An A-type procyanidin as defined above may be derivatized, for instanceesterified, at one or more of the OH groups on one or more of theconstituent flavan-3-ol units. A given flavan-3-ol unit may thuscomprise one or more ester groups, preferably gallate ester groups, atone or more of the 3-, 5-, 7-, 3′- and 4′-ring positions. It may inparticular be a mono-, di-, tri-, tetra- or penta-gallated unit.

Examples of the compounds useful for products, and in the methods of thepresent invention, include the compounds wherein the integer n is 3 to12; 4 to 12; 5 to 12; 4 to 10; or 5 to 10. In some embodiments, n is 2to 4, or 2 to 5, for example n is 2 or 3.

In one embodiment, the A-type procyanidin is epicatechin-(4β→8;2β→O→7)-catechin (i.e., A1 dimer), or a pharmaceutically acceptable saltor derivative thereof, and has the following formula:

In another embodiment, the A-type procyanidin is epicatechin-(4β→8;2β→O→7)-epicatechin (i.e., A2 dimer) and has the following formula:

In yet another embodiment, the A-type procyanidin is an A-type trimerand has the following formula:

A-type procyanidins may be of natural origin or synthetically prepared.For example, A-type procyanidins may be isolated from peanut skins asdescribed in Example 1, or as described in Lou et al., Phytochemistry,51: 297-308 (1999), or Karchesy and Hemingway, J. Agric. Food Chem.,34:966-970 (1986), the relevant portions of each being herebyincorporated herein by reference. Mature red peanut skin contain about17% by weight procyanidins, and among the dimeric procyanidinsepicatechin-(4β→8; 2β→O→7)-catechin dominates, with smaller proportionof epicatechin-(4β→8; 2β→O→7)-epicatechin being present. However, inaddition to procyanidins having (4β→8; 2β→O→7) double linkages,procyanidins having (4→6; 2→O→7) double linkages are also found inpeanut skins.

Other sources of the above compounds are cranberries as described, forexample in Foo et al., J Nat. Prod., 63: 1225-1228, and in Prior et al.,J Agricultural Food Chem., 49(3):1270-76 (2001), the relevant portionsof each being hereby incorporated herein by reference. Other sourcesinclude Ecdysanthera utilis (Lie-Chwen et al., J Nat. Prod., 65:505-8(2002)) and Aesculus hippocastanum (U.S. Pat. No. 4,863,956), therelevant portions of each being hereby incorporated herein by reference.

A-type compounds may also be obtained from B-type procyanidins viaoxidation using 1,1-diphenyl-2-pycrylhydrazyl (DPPH) radicals underneutral conditions as described in Kondo et al., Tetrahedron Lett., 41:485 (2000), the relevant portions of which are hereby incorporatedherein by reference. Methods of obtaining natural and synthetic B-typeprocyanidins are well known in the art and are described, for example,in U.S. Pat. Nos. 6,670,390 to Romanczyk et al.; 6,207,842 to Romanczyket al.; 6,420,572 to Romanczyk et al.; and 6,156,912 to Romanczyk et al.

The A-type procyanidins may be used in the compositions described hereinand administered in the form of an extract (e.g. peanut skins extract)comprising A-type procyanidins as the main component.

The A-type procyanidins may be isolated and purified, i.e., they areseparated from compounds with which they naturally occur (if the A-typeprocyanidin is of natural origin), or they are synthetically prepared,in either case such that the level of contaminating compounds(impurities) does not significantly contribute to, or detract from, theeffectiveness of the A-type procyanidin. For example, an isolated andpurified A1 dimer is separated from A2 dimer, with which it may occur innature, to the extent achievable by the available commercially viablepurification and separation techniques.

The compounds may, be substantially pure, i.e., they possess the highestdegree of homogeneity achievable by the available purification,separation and/or synthesis technology. As used herein, a “substantiallypure A1 dimer” is separated from A2 dimer to the extent technologicallyand commercially possible, and a “substantially pure A-type trimer” isseparated from other A-type oligomers (to the extent technologically andcommercially possible) but may contain a mixture of several A-typetrimers. In other words, the phrase “isolated and purified trimer”refers primarily to one trimer, while a “substantially pure trimer” mayencompass a mixture of trimers.

In some embodiments, the A-type procyanidins are at least 80% pure,preferably at least 85% pure, at least 90% pure, at least 95% pure, atleast 98% pure, or at least 99% pure. Such compounds are particularlysuitable for pharmaceutical applications.

Methods of Use

Any compound described in the application may be used to practice themethods described herein. As shown in Example 3, A-type procyanidinsaffect the nitric oxide (NO) pathway in endothelial cells helpingpreserve the NO pool. Without being bound by theory, the NO pool ispreserved by inducing NO synthesis and/or decreasing NO degradation. Thecompounds also cause vasorelaxation of constricted blood vessels. Theymay also be used for anti-platelet therapy as shown in Example 4.

Thus, the invention relates to a method of treating or preventing anNO-responsive disease or disorder by administering to a subject in needthereof an effective amount of an A-type procyanidin oligomer composedof n monomeric, flavan-3-ol units of the formula:

wherein

-   (i) the monomeric units are connected via interflavan linkages 4→6    and/or 4→8;-   (ii) at least two of the monomeric units are additionally linked by    an A-type interflavan linkage (4→8; 2→O→7) or (4→6; 2→O→7);-   (iii) n is 2 to 12;    or a pharmaceutically acceptable salt or derivative thereof,    and wherein the subject is a human or a veterinary animal.

The A-type procyanidins may be isolated and purified or substantiallypure. In some embodiments, the above compounds may be at least about 80%pure, at least about 85% pure, at least about 90% pure, at least 95%pure or at least 98% pure. Examples of the compounds for use in theabove method include the compounds wherein the integer n is 3 to 12; 4to 12; 5 to 12; 4 to 10; or 5 to 10. In some embodiments, n is 2 to 4,or 2 to 5, for example n is 2 or 3.

As used herein, an “NO-responsive disease or disorder” refers to ahealth condition which responds to treatment with NO. Examples of suchconditions include, but are not limited, to NO-mediated or NO-dependentdiseases and disorders, in which the pathology of the disease/disorderis caused by abnormal functioning of the NO pathway. Preferably theconditions include hypertension, cardiovascular disease, coronary arterydisease and/or vascular circulation disorders, heart attack, stroke,congestive heart failure, kidney failure, and renal disease. The A-typeprocyanidins may be administered alone or in combination with anothercardiovascular therapeutic agent.

Because high blood pressure increases the risk of heart attack, stroke,congestive heart failure, and kidney failure, A-type procyanidins whichcause vasorelaxation can be utilized, alone or in combination with othercardiovascular-protective agents, to prevent these conditions.Particularly suitable subjects include subjects with high blood pressurein combination with diabetes, obesity, high cholesterol levels and/orsmokers, in which patients the risk of heart attack and stroke increasesseveral times.

As used herein, “treatment” means improving an existing medicalcondition, such as cardiovascular disease, for example by slowing downthe disease progression, prolonging survival, reducing the risk ofdeath, and/or providing a measurable improvement of disease parameters.

The term “preventing” means reducing the risks associated withdeveloping a disease, including reducing the onset of the disease.

As used herein, the terms “cardiovascular-protective or therapeuticagent” refers to an agent other than A-type procyanidin which iseffective to treat or protect cardiovascular system. Examples of suchagents are anti-platelet therapy agents (e.g. COX inhibitors, such asaspirin; B-type procyanidins); NO-modulating agents, cholesterolreducing agents (e.g. sterol, stanol).

In some embodiments, the invention relates to the following methods:

A method of treating or preventing an NO-responsive disease or disorderby administering to a subject in need thereof an effective amount ofepicatechin-(4β→8; 2β→O→7)-catechin (i.e., A1 dimer) of the followingformula, or a pharmaceutically acceptable salt or derivative thereof:

wherein the subject is a human or a veterinary animal. The A1 dimer maybe isolated and purified. In some embodiments, the above compound may beat least about 90% pure, at least 95% pure or at least 98% pure.

A method of treating or preventing an NO-responsive disease or disorderby administering to a subject in need thereof an effective amount ofepicatechin-(4β→8; 2β→O→7)-epicatechin (i.e., A2 dimer) of the followingformula, or a pharmaceutically acceptable salt or derivative thereof:

wherein the subject is a human or a veterinary animal. The A2 dimer maybe isolated and purified. In some embodiments, the above compound may beat least about 90% pure, at least 95% pure or at least 98% pure.

A method of treating or preventing an NO-responsive disease or disorderby administering to a subject in need thereof an effective amount of anA-type trimer of the following formula, or a pharmaceutically acceptablesalt or derivative thereof:

wherein the subject is a human or a veterinary animal. The A-type trimermay be isolated and purified, or substantially pure. In someembodiments, the above compounds may be at least about 80% pure, atleast about 85% pure, at least about 90% pure, at least 95% pure or atleast 98% pure.

In certain embodiment, the invention provides for the followingexemplary methods:

A method of treating hypertension by administering to a subject in needthereof an effective amount of an A-type procyanidin oligomer composedof n monomeric, flavan-3-ol units of the formula, or a pharmaceuticallyacceptable salt or derivative thereof:

wherein

-   (i) the monomeric units are connected via interflavan linkages 4→6    and/or 4→8;-   (ii) at least two of the monomeric units are additionally linked by    an A-type interflavan linkage (4→8; 2→O→7) or (4→6; 2→O→7);-   (iii) n is 2 to 12;    and wherein the subject is a human or a veterinary animal.

The A-type procyanidins may be isolated and purified or substantiallypure. In some embodiments, the above compounds may be at least about 80%pure, at least about 85% pure, at least about 90% pure, at least 95%pure or at least 98% pure.

Examples of the compounds for use in the above method include thecompounds wherein the integer n is 3 to 12; 4 to 12; 5 to 12; 4 to 10;or 5 to 10. In some embodiments, n is 2 to 4, or 2 to 5, for example nis 2 or 3.

In certain embodiments, the invention includes the following exemplarymethods:

A method of treating hypertension by administering to a subject in needthereof an effective amount of epicatechin-(4β→8; 2β→O→7)-catechin(i.e., A1 dimer) of the formula, or a pharmaceutically acceptable saltor derivative thereof:

wherein the subject is a human or a veterinary animal. The A1 dimer maybe isolated and purified. In some embodiments, the above compound may beat least about 90% pure, at least 95% pure or at least 98% pure.

A method of treating hypertension by administering to a subject in needthereof an effective amount of epicatechin-(4β→8; 2β→O→7)-epicatechin(i.e., A2 dimer) of the following formula, or a pharmaceuticallyacceptable salt or derivative thereof:

wherein the subject is a human or a veterinary animal. The A2 dimer maybe isolated and purified. In some embodiments, the above compound may beat least about 90% pure, at least 95% pure or at least 98% pure.

A method of treating hypertension by administering to a subject in needthereof an effective amount of A-type trimer of the following formula,or a pharmaceutically acceptable salt or derivative thereof:

wherein the subject is a human or a veterinary animal. The A-typeprocyanidin trimer may be isolated and purified or substantially pure.In some embodiments, the above compounds may be at least about 80% pure,at least about 85% pure, at least about 90% pure, at least 95% pure orat least 98% pure.

Also within the scope of the invention are methods of improving/treatingerectile dysfunction in a person in need thereof using any of thecompounds recited herein.

The effective amount for use in the above methods may be determined by aperson of skill in the art using the guidance provided herein andgeneral knowledge in the art. For example, the effective amount may besuch as to achieve a physiologically relevant concentration in the body(e.g. blood) of a mammal. Such a physiologically relevant concentrationmay be at least about 10 nanomolar (nM), preferably at least about 20nM, or at least about 100 nM, and more preferably at least about 500 nM.In one embodiment, at least about one micromole in the blood of themammal, such as a human, is achieved. The compounds of formula A_(n), asdefined herein, may be administered at from about 50 mg/day to about1000 mg/day, preferably from about 100-150 mg/day to about 900 mg/day,and most preferably from about 300 mg/day to about 500 mg/day. However,amounts higher than stated above may be used.

The compounds may be administered acutely, or treatments/preventiveadministration may be continued as a regimen, i.e., for an effectiveperiod of time, e.g., daily, monthly, bimonthly, biannually, annually,or in some other regimen, as determined by the skilled medicalpractitioner for such time as is necessary. The administration may becontinued for at least a period of time required to exhibittherapeutic/prophylactic effects. Preferably, the composition isadministered daily, most preferably two or three times a day, forexample, morning and evening to maintain the levels of the effectivecompounds in the body of the mammal. To obtain the most beneficialresults, the composition may be administered for at least about 30, orat least about 60 days. These regiments may be repeated periodically.

Compositions and Formulations

The compounds of the invention may be administered as a pharmaceutical,food, food additive or a dietary supplement.

As used herein a “food” is a material consisting essentially of protein,carbohydrate and/or fat, which is used in the body of an organism tosustain growth, repair and vital processes and to furnish energy. Foodsmay also contain supplementary substances such as minerals, vitamins andcondiments. See Merriam-Webster's Collegiate Dictionary, 10th Edition,1993. The term food includes a beverage adapted for human or animalconsumption. As used herein a “food additive” is as defined by the FDAin 21 C.F.R. 170.3(e)(1) and includes direct and indirect additives. Asused herein, a “pharmaceutical” is a medicinal drug. SeeMerriam-Webster's Collegiate Dictionary, 10th Edition, 1993. Apharmaceutical may also be referred to as a medicament. As used herein,a “dietary supplement” is a product (other than tobacco) that isintended to supplement the diet that bears or contains the one or moreof the following dietary ingredients: a vitamin, a mineral, an herb orother botanical, an amino acid, a dietary substance for use by man tosupplement the diet by increasing the total daily intake, or aconcentrate, metabolite, constituent, extract or combination of theseingredients.

Pharmaceuticals containing the inventive compounds, optionally incombination with another cardiovascular-protective or therapeutic agent,may be administered in a variety of ways such as orally, sublingually,bucally, nasally, rectally, intravenously, parenterally and topically. Aperson of skill in the art will be able to determine a suitable mode ofadministration to maximize the delivery of A-type procyanidins,optionally in combination with another cardiovascular-protective ortherapeutic agent. Thus, dosage forms adapted for each type ofadministration are within the scope of the invention and include solid,liquid and semi-solid dosage forms, such as tablets, capsules, gelatincapsules (gelcaps), bulk or unit dose powders or granules, emulsions,suspensions, pastes, creams, gels, foams, jellies or injection dosageforms. Sustained-release dosage forms are also within the scope of theinvention and may be prepared as described in U.S. Pat. Nos. 5,024,843;5,091,190; 5,082,668; 4,612,008 and 4,327,725, relevant portions ofwhich are hereby incorporated herein by reference. Suitablepharmaceutically acceptable carriers, diluents, or excipients aregenerally known in the art and can be determined readily by a personskilled in the art. The tablet, for example, may comprise an effectiveamount of the A-type procyanidin containing composition and optionally acarrier, such as sorbitol, lactose, cellulose, or dicalcium phosphate.

The dietary supplement containing A-type procyanidins, and optionallyanother cardiovascular-protective or therapeutic agent, may be preparedusing methods known in the art and may comprise, for example, nutrientsuch as dicalcium phosphate, magnesium stearate, calcium nitrate,vitamins, and minerals.

Further within the scope of the invention is an article of manufacturesuch as a packaged product comprising the composition of the invention(e.g. a food, a dietary supplement, a pharmaceutical) and a labelindicating the presence of, or an enhanced content of the inventivecompounds or directing use of the composition for methods describedherein.

Also within the scope of the invention is an article of manufacture(such as a packaged product or kit) adapted for use in combinationtherapy comprising at least one container and at least one A-typeprocyanidin, or a pharmaceutically acceptable salt or derivativesthereof. The article of manufacture further comprises at least oneadditional agent, a cardiovascular-protective or therapeutic agent(i.e., other than the A-type procyanidin, or a pharmaceuticallyacceptable salt or derivative thereof), which agent may be provided as aseparate composition, in a separate container, or in admixture with thecompound of the invention.

As described above, cardiovascular-protective or therapeutic agents areeffective to treat or protect cardiovascular system. Examples of suchagents are anti-platelet therapy agents (e.g. COX inhibitors, such asaspirin); NO-modulating agents, cholesterol reducing agents (e.g.sterol, stanol).

In certain embodiments, cardiovascular-protective or therapeutic agentsoptionally administered with A-type procyanidins may be B-typeprocyanidins, for example cocoa flavanols and/or procyanidins asdescribed below.

The B-type polyphenols for use in the present invention may be ofnatural origin, for example, derived from a cocoa bean or anothernatural source of polyphenols, or prepared synthetically. For example,the B-type procyanidins and their derivatives are those described inU.S. Pat. No. 6,670,390 to Romanczyk et al., the relevant portions ofwhich are hereby incorporated herein by reference. A person of skill inthe art may select natural or synthetic polyphenol based on availabilityor cost. Polyphenols may be included in the composition in the form of acocoa ingredient containing cocoa polyphenols, for example, chocolateliquor included in chocolate, or may be added independently of cocoaingredients, for example, as an extract, extract fraction, isolated andpurified individual compound, pooled extract fractions or asynthetically prepared compound.

The term “cocoa ingredient” refers to a cocoa solids-containing materialderived from shell-free cocoa nibs such as chocolate liquor andpartially or fully-defatted cocoa solids (e.g. cake or powder).

The B-type procyanidin oligomers may have from 2 to about 18, preferablyfrom 2 to about 12, and most preferably from 2 to about 10 monomericunits. Alternatively, the oligomers may have from 3-18, preferably 3-12,and more preferably 3-10 monomeric units; or from 5-18, preferably 5-12and more preferably 5-10 monomeric units. For example, oligomers may bedimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers,nonamers and decamers. In the oligomer, monomers are connected viainterflavan linkages of (4→6) and/or (4→8). Oligomers with exclusively(4→8 linkages are linear; while the presence of at least one (4→6) bondresults in a branched oligomer. Also within the scope of the inventionare oligomers comprising at least one non-natural linkage (6→6), (6→8),and (8→8). The synthesis of such non-naturally occurring oligomers isdescribed in the International Appl. No. PCT/US00/08234 published onOct. 19, 2000 as WO 00/61547, the relevant portions of which are herebyincorporated herein by reference.

The B-type procyanidins may be prepared by extraction from cocoa beans,cocoa nibs, or cocoa ingredients such as chocolate liquor, partiallydefatted cocoa solids, and/or fully defatted cocoa solids. Preferably,the extract is prepared from a fully or partially defatted cocoa powder.Beans from any species of Theobroma, Herrania or inter- andintra-species crosses thereof may be used. The extract may be preparedfrom fermented, underfermented or unfermented beans, the fermented beanshaving the least amount of cocoa polyphenols and the unfermented themost. The selection of beans may be made based on the fermentationfactor of the beans, for example, the extract may be made from the beanshaving a fermentation factor of about 275 or less. Optimizing the levelof polyphenols in the cocoa ingredient and extract thereof bymanipulating the degree of fermentation may be done as described in theInternational Appl. No. PCT/US97115893 published as W098/09533, therelevant portions of which are hereby incorporated herein by reference.

Cocoa polyphenols may be extracted from cocoa ingredients that have beenprocessed using traditional methods of cocoa processing (described, forexample, in Industrial Chocolate Manufacture and Use, ed. Beckett, S.T., Blackie Acad. & Professional, New York, 1997, such as in Chapters 1,5 and 6) or using an improved processing method described in U.S. Pat.No. 6,015,913 to Kealey et al. that preserves polyphenols (by preventingtheir destruction) in cocoa ingredients in contrast to the traditionalmethods. The improved cocoa processing method omits the traditionalroasting step. Thus, cocoa ingredients obtainable by (a) heating thecocoa bean for a time and a temperature sufficient to loosen the cocoashell without roasting the cocoa nib; (b) winnowing the cocoa nib fromthe cocoa shell; (c) screw pressing the cocoa nib and (d) recovering thecocoa butter and partially defatted cocoa solids which contain preservedlevels of cocoa polyphenols, may be used. The method retains a muchhigher level of higher procyanidin oligomers than traditional processingmethods. Cocoa solids produced by this method may contain greater than20,000 μg of total flavanol and/or procyanidins per gram nonfat solids;preferably greater than 25,000 μg/g, more preferably greater than 28,000μg/g, and most preferably greater than 30,000 μg/g. For purposes of thisinvention, the total flavanol and/or procyanidin amounts are determinedas described in Example 2.

B-type procyanidins may be extracted from the sources indicated above,or any other polyphenol or flavanol or procyanidin containing source,using solvents in which the polyphenols dissolve. Suitable solventsinclude water or organic solvent such as methanol, ethanol, acetone,isopropyl alcohol and ethyl acetate. Solvent mixtures may also be used.When water is used as the solvent, it may be slightly acidified, forexample with acetic acid. Examples of some solvents are mixtures ofwater and organic solvent, for example aqueous methanol, ethanol oracetone. Aqueous organic solvents may contain, for example, from about50% to about 95% of organic solvent. Thus, about 50%, about 60%, about70%, about 80% and about 90% organic solvent in water may be used. Thesolvent may also contain a small amount of acid such as acetic acid, forexample, in the amount of about 0.5% to about 1.0%. The composition ofthe extracts, i.e., the representation (i.e., oligomeric profile) andthe amount of procyanidin oligomers, will depend on the choice ofsolvents. For example, the water extract contains primarily monomers,the ethyl acetate extract contains monomers and lower oligomers, mainlydimers and trimers, and the aqueous methanol, ethanol or acetone extractcontains monomers and a range of higher oligomers. One of the solventsfor extraction of monomer as well as higher procyanidin oligomers isabout 70% acetone. However, any extract containing polyphenols is usefulin the invention. The methods of cocoa polyphenol extraction are knownin the art and are described, for example, in the U.S. Pat. No.5,554,645 to Romanczyk et al. and the International Appl. No.PCT/US97/05693, published as W097/36497. Thus, in one embodiment, thecocoa extract is prepared by reducing cocoa beans to cocoa powder,defatting the powder, extracting the cocoa polyphenols, and purifyingthe extract. The cocoa powder can be prepared by freeze-drying the cocoabeans and pulp, depulping and dehulling the freeze-dried cocoa beans,and grinding the dehulled beans.

The B-type cocoa polyphenol extract may be purified, for example, byremoval of the caffeine and/or theobromine, and further purified by gelpermeation chromatography and/or High Pressure Liquid Chromatography(HPLC). Gel permeation chromatography (e.g. on Sephadex LH-20) may beused to enrich the extract for higher procyanidin oligomers. Forexample, the eluate containing monomers and lower oligomers may not becollected until the oligomer(s) of choice begins eluting from thecolumn. An example of such an extract is known in the art and isdescribed in Example 5 of the International Appl. No. PCT/US97/05693,published as W097/36497, the relevant portions of which are herebyincorporated by reference herein. By using preparative HPLC, forexample, normal phase HPLC, the extract may be fractionated, forexample, into monomeric and oligomeric fractions containing at least 50%by weight of the monomer or specific oligomer(s). When a particularfraction contains the monomers or any of the lower oligomers (e.g.dimers, trimers or tetramers fraction), the fraction contain about 90 to95% by weight of the particular oligomeric fraction. The desiredfractions may be pooled after separation to obtain a combination ofoligomers of choice for example to contain oligomers 3-10 or 5-10. Aperson of skill in the art can manipulate the chromatographic conditionsto achieve the desired procyanidin profile in view of the guidance inthis specification, general knowledge in the art and, for example, theteachings of U.S. Pat. No. 5,554,645 to Romanczyk et al. and theInternational Appl. No. PCT/US97/05693, published as W097/36497.

The monomeric fraction typically contains a mixture of monomersepicatechin and catechin; and the oligomeric fraction typically containsa mixture of dimers (in a dimer fraction), trimers (in a trimerfraction), tetramers (in a tetramer fraction), etc. Mixtures of monomersand oligomers occur in isolated fractions because cocoa contains morethan one type of each of monomer, dimer, etc. The oligomeric variabilityoccurs as a result of two monomers, epicatechin and catechin, that arebuilding blocks of procyanidins, as well as the chemical bond connectingmonomers in the oligomer. Thus, cocoa dimers are primarily B2 and B5,each of which contains two monomers of epicatechin. Individual monomersand oligomers may be obtained using reversed-phase HPLC, e.g. using aC18 column.

B-type cocoa polyphenol may be used in the compositions of the inventionas a cocoa extract, e.g. solvent-derived extract, cocoa fraction,isolated compounds or in the form of a cocoa ingredient or a chocolatecontaining an effective amount of cocoa flavanols and/or procyanidins.The cocoa ingredients may be prepared using traditional cocoa processingprocedures but is preferably prepared using the method described in U.S.Pat. No. 6,015,913 to Kealey et al. Alternatively, to enhance the levelof cocoa polyphenols, chocolate liquor and cocoa solids prepared fromcocoa beans having a fermentation factor of about 275 or less may beused. These ingredients have cocoa polyphenol content that is higherthan can be obtained using traditional cocoa processing methods (e.g.with roasting) and fully fermented beans. The chocolate may be preparedusing conventional techniques from the ingredients described above orusing an improved process for preserving cocoa polyphenols duringchocolate manufacturing as described in the International Appl. No.PCT/US99/05414 published as W099/45788, the relevant portions of whichare hereby incorporated herein by reference. A chocolate prepared by atleast one of the following non-traditional processes is referred toherein as a “chocolate having a conserved amount of cocoa polyphenols”:(i) preparing cocoa ingredients from underfermented or unfermented cocoabeans; (ii) preserving cocoa polyphenol during cocoa ingredientmanufacturing process; and (iii) preserving cocoa polyphenol duringchocolate manufacturing process.

Synthetic B-type procyanidins may also be used and are prepared bymethods known in the art and as described for example in theInternational Appl. No. PCT/US98/21392 published as W099/19319, therelevant portions of which are hereby incorporated herein by reference.

Flavanol and/or procyanidin derivatives may also be useful. Theseinclude esters of monomer and oligomers such as the gallate esters (e.g.epicatechin gallate and catechin gallate); compounds derivatized with asaccharide moiety such as mono- or di-saccharide moiety (e.g.β-D-glucose), glycosylated monomers and oligomers, and mixtures thereof;metabolites of the procyanidin monomers and oligomers, such as thesulphated, glucouronidated, and methylated forms except for the enzymecleavage products of procyanidins generated by colonic microflorametabolism. The derivatives may be from natural sources or preparedsynthetically.

The foods comprising A-type and/or B-type procyanidins and optionallyanother cardiovascular-protective/treatment agent may be adapted forhuman or veterinary use, and include pet foods. The food may be otherthan a confectionery, however, the preferred cholesterol lowering foodis a confectionery such as a standard of identity (SOI) and non-SOIchocolate, such as milk, sweet and semi-sweet chocolate including darkchocolate, low fat chocolate and a candy which may be a chocolatecovered candy. Other examples include a baked product (e.g. brownie,baked snack, cookie, biscuit) a condiment, a granola bar, a toffee chew,a meal replacement bar, a spread, a syrup, a powder beverage mix, acocoa or a chocolate flavored beverage, a pudding, a rice cake, a ricemix, a savory sauce and the like. If desired, the foods may be chocolateor cocoa flavored. Food products may be chocolates and candy bars, suchas granola bars, containing nuts, for example, peanuts, walnuts,almonds, and hazelnuts. It should be noted that the addition of nutswith skins to the food described herein may also increase the totalpolyphenol content since, for example, peanut skins contain about 17%flavanols and procyanidins and almond skins contain about 30% flavanolsand procyanidins. Ground peanut skins may be added to the compositionsof the invention. In one embodiment, the nut skins, e.g. peanut skins,are added to the nougat of a chocolate candy.

In certain embodiments, the non-chocolate food product contains fromabout at least 5 micrograms/g to about 10 mg/g, and, for example, atleast 5 micrograms/g food product, preferably at least 10 microgram/g,more preferably at least 100 micrograms/g of flavanols and/or B-typeprocyanidins and/or A-type procyanidins. If desired, the non-chocolatefood products can contain much higher levels of cocoa procyanidins thanthose found in the chocolate food products described below.

The chocolate confectionery may be milk or dark chocolate. In certainembodiments, the chocolate comprises at least 3,600 micrograms,preferably at least 4,000 micrograms, preferably at least 4,500micrograms, more preferably at least 5,000 micrograms, and mostpreferably at least 5,500 micrograms of flavanols and/or B-typeprocyanidins and/or A-type procyanidins each per gram of chocolate,based on the total amount of nonfat cocoa solids in the product. Inother embodiments, the chocolate contains at least 6,000 micrograms,preferably at least 6,500 micrograms, more preferably at least 7,000micrograms, and most preferably at least 8,000 micrograms of flavanolsand/or B-type procyanidins and/or A-type procyanidins per gram, and evenmore preferably 10,000 micrograms/g based on the nonfat cocoa solids inthe product.

A milk chocolate confectionery may have at least 1,000 micrograms,preferably at least 1,250 micrograms, more preferably at least 1,500micrograms, and most preferably at least 2,000 micrograms flavanolsand/or B-type procyanidins and/or A-type procyanidins each per gram ofmilk chocolate, based on the total amount of nonfat cocoa solids in themilk chocolate product. In the preferred embodiment, the milk chocolatecontains at least 2,500 micrograms, preferably at least 3,000micrograms, more preferably at least 4,000 micrograms, and mostpreferably at least 5,000 micrograms flavanols and/or B-typeprocyanidins and/or A-type procyanidins each per gram of milk chocolate,based on the total amount of nonfat cocoa solids in the milk chocolateproduct.

L-arginine may be added to food products in the amount that can vary.Typically, cocoa contains between 1 to 1.1 grams of L-arginine per 100grams of partially defatted cocoa solids. It can range from 0.8 to 1.5per 100 grams of cocoa. In some embodiments, the chocolate food productsof this invention contain L-arginine in an amount greater than thatwhich naturally occurs in the cocoa ingredients. Knowing the amount ofcocoa ingredients and L-arginine used in the food product, one ofordinary skill in the art can readily determine the total amount ofL-arginine in the final product. The food product will generally containat least 5 micrograms/g, preferably at least 30 micrograms/g, or atleast 60 micrograms/g, even more preferably at least 200 micrograms/gfood product.

A daily effective amount of flavanols and/or A-type and/or B-typeprocyanidins may be provided in a single serving. Thus, a confectionery(e.g. chocolate) may contain at least about 100 mg/serving (e.g.150-200, 200-400 mg/serving).

The invention is further described in the following non-limitingexamples.

EXAMPLES Example 1 Extraction and Isolation of A-type Procyanidins

Extraction

Finely ground peanut skins (498 g) were defatted with hexane (2×2000mL). Hexane was removed by centrifugation at ambient temperature, 5 minat 3500 rpm, and discarded. Residual hexane was allowed to evaporateovernight. The following day, defatted peanut skins were extracted for 2hours at ambient temperature with acetone:water:acetic acid (70:29.5:0.5v/v/v) (2×2000 mL). Extracts were recovered by centrifugation (ambienttemperature, 5 min at 3500 rpm). Organic solvents were removed by rotaryevaporation under partial pressure (40° C.). Aqueous portion ofextraction solvent was removed by freeze drying to provide a brown-redcrusty solid (51.36 g).

Gel Permeation of Crude Peanut Skin Extract

Crude peanut skin extract (24 g), obtained as described above, wasdissolved in 70% methanol (150 mL), refrigerated for 1 hour, vortexedfor 3 sec, then centrifuged at ambient temperature, for 5 min at 3500rpm. The supernatant was loaded atop a large column containing SephadexLH-20 (400 g) preswollen in methanol. Column was eluted isocraticallywith 100% methanol at a flow rate of 10 mL/min. Twenty nine fractions,250 mL each, were collected and combined in accordance to theircomposition as determined by NP-HPLC (Adamson et al., J. Ag. Food Chem.,47: 4184-4188, 1999) to give a total of eight fractions (i-viii).Fraction i contained monomers epicatechin and catechin, fraction ii-viicontained dimers, trimers or mixtures thereof. Fraction v (1.8 g) andvii (2.7 g) contained a preponderance of dimers and trimers,respectively, and were selected for further purification.

Purification of A-type Dimers and Trimers

Fraction v (1.8 g) was dissolved in 0.1% acetic acid in 20% methanol (40mg/mL). Injection volumes were 2 mL. Separations were conducted on aHypersil ODS (250×23 mm) under gradient conditions. Mobile phasesconsisted of 0.1% acetic acid in water (mobile phase A) and 0.1% aceticacid in methanol (mobile phase B). Gradient conditions were: 0-10 min,20% B isocratic; 10-60 min, 20-40% B linear; 60-65 min, 40-100% Blinear. Separations were monitored at 280 nm. Fractions with equalretention times from several preparative separations were combined,rotary evaporated at 40° C. under partial vacuum and freeze dried. Fivefractions (a-e) were obtained. Fractions d and e were characterized byLCMS as dimers A1 and A2, respectively. In addition to A1 and A2 dimers,four different dimers were previously isolated from peanut skins (Lou etal., Phytochemistry 51, 297-308, 1999).

Fraction vii was purified as described above to obtain a single trimerwith an A-linkage having the formula represented above.

The structures of purified compounds were confirmed by MassSpectroscopy, and the purity of the compounds was determined using HPLCat UV 280 nm. A1 dimer was 95% pure, A2 dimer was 91% pure, and A trimerwas 84% pure.

Example 2 Determination of Flavanols/Procyanidins

Procyanidins were quantified as follows: a composite standard was madeusing commercially available (−)-epicatechin, and dimers throughdecamers obtained in a purified state by the methods described inHammerstone, J. F. et al., J. Ag. Food Chem.; 1999; 47 (10) 490-496;Lazarus, S. A. et al., J. Ag. Food Chem.; 1999; 47 (9); 3693-3701; andAdamson, G. E. et al., J. Ag. Food Chem.; 1999; 47 (10) 4184-4188.Standard Stock solutions using these compounds were analyzed using thenormal-phase HPLC method described in the previously cited Adamsonreference, with fluorescence detection at excitation and emissionwavelengths of 276 nm and 316 nm, respectively. Peaks were grouped andtheir areas summed to include contributions from all isomers within anyone class of oligomers and calibration curves were generated using aquadratic fit. Monomers and smaller oligomers had almost linear plotswhich is consistent with prior usage of linear regression to generatemonomer-based and dimer-based calibration curves.

These calibration curves were then used to calculate procyanidin levelsin samples prepared as follows: First, the cocoa or chocolate sample(about 8 grams) was defatted using three hexane extractions (45 mLeach). Next, one gram of defatted material was extracted with 5 mL ofthe acetone/water/acetic acid mixture (70:29.5:0.5 v/v). The quantity ofprocyanidins in the defatted material was then determined by comparingthe HPLC data from the samples with the calibration curves obtained asdescribed above (which used the purified oligomers). The percentage offat for the samples (using a one gram sample size for chocolate orone-half gram sample size for liquors) was determined using astandardized method by the Association of Official Analytical Chemists(AOAC Official Method 920.177). The quantity of total procyanidin levelsin the original sample (with fat) was then calculated. Calibration wasperformed prior to each sample run to protect against column-to-columnvariations.

Example 3 Effect of A-type Procyanidins on NO Production andVasorelaxation

The compounds obtained as described in Example 1, were investigated fortheir effect on nitric oxide (NO) production and vasorelaxation usingserum-free human umbilical vein endothelial cell (HUVEC) culture systemin vitro and rabbit aortic ring model ex vivo, respectively. NOproduction by endothelial cells and relaxation of pre-constricted aorticrings are two main markers for evaluating cardiovascular effects of testcompounds.

In Vitro Experiment

HUVECs obtained from a single donor were cultured in serum free, lowprotein (0.5 g/l), antibiotic-free cell culture medium supplemented withessential growth factors, nutrients and minerals. The cultured cellexpressed endothelial markers (von Willebrand factor, CD31 antigen,uptake of Dil-Ac-LDL) and exhibited the typical “cobble-stonemorphology” when grown to confluence. The cell culture medium wassubstituted with apo-transferrin, superoxide dismutase, and catalase toexclude secondary effects of test compounds involving theirauto-oxidation mediated hydrogen peroxide formation.

Test compounds were evaluated with respect to their potential to acutely(2 hours) and chronically (5 doses given in a 24 h period) modulate NOproduction. Positive controls (acetylcholine and/or histamine) andnegative control L-NNMA (NO synthase inhibitor) were included in allexperiments. Cell counts and total protein were used to assessintra-assay variation. Potential toxic effects of tested compounds werealso monitored (MTT reduction was measured).

NO production was evaluated by measuring the total amount of all majornitric oxide end products (NOx, including nitrate, nitrite,nitrosothiols) present in the cell culture medium. For this purpose NOxwere directly reduced by vanadium(III)chloride/HCl at 95 degrees C.yielding NO. The amount of NO released from the culture medium wassubsequently evaluated by measuring the chemiluminescence emitted duringthe stoichiometrical reaction between ozone and NO using NO Analyzer(Sievers Instruments, Inc. Boulder, Colo.).

The data presented herein were obtained from three experiments and wereexpressed as the concentration of NO present (in μmol/l) (as NOx) in thecell culture medium +/−standard deviation (SD). The data were correctedfor the NOx intrinsically present in the fully supplemented cell culturemedium and normalized with respect of the volume of media from which theample was drawn. Data were analyzed using Student's t-test with a 95%level of confidence. P values equal to or less than 0.005 were definedas statistically significant.

For the acute effect test, HUVECs were incubated with a single dose ofA2 dimer and A-type trimer for 2 and 24 hours at concentrations of 100nM, 1 μM, and 10 μM at 37 C and 5% C0₂. No effect was observed on NOproduction after 2 hours. After 24 hours following a single dose, noeffect was seen at 1 μM concentration. Both test compounds increased NOproduction above the control level at concentration of 10 μM (of thetwo, A2 performed better) but neither to statistically significantlevels. Based on the MTT assay, the test compounds did not have toxiceffects.

For the chronic effect test, HUVECs were incubated with 5 subsequentdoses of test compounds, each for 24 hours. After each 24 hourtreatment, culture medium was replaced. A2 dimer and A-type trimer weretested. A-type trimer exhibited statistically significant increase in NOproduction (p=0.041).

Ex Vivo Experiment

Effect of A-type procyanidins on endothelium-dependent relaxation wastested in an ex vivo experiment performed as previously described byKarim et al., J. Nutrl Suppl., 130 (8S): 2105S-2108S (2000), therelevant portions of which are hereby incorporated herein by reference.The advantage of using this method is that it assesses functionalcardiovascular end points. The method is only able to assess acuteevents and does not allow for the identification of drug-induced proteinexpression/activity.

In summary, rabbit aortic rings were obtained from male New ZealandWhite rabbits. Following isolation, the rings were mounted in oxygenatedKreb's buffer, and pre-constricted with NE (10⁻⁶ M). When the tensionhad reached a steady state, cumulative concentrations of the testcompounds were applied (10-9 to 10-4 M).

A positive control acetylcholine (10⁻⁶M) and a negative control L-NAMEwere included in the experiment. Use of L-NAME, which is a NO synthase(NOS) inhibitor, allows for differentiating between endotheliumdependent and endothelium independent relaxation events. Denuding ofaortic rings represent a similar control. 400 U/mL of catalase was addedinto the aortic bath prior to the addition of each of the test compoundsto ensure that the observed effects are not caused by hydrogen peroxide(H₂0₂) generation in the culture medium. The relaxation response wasmeasured as a function of the decrease in the tension (g) exerted by theaortic rings over time. Data obtained were expressed as a percentrelaxation of the norepinephrine (NE) constricted rings. The samestatistical approach as described above was used. Dose response curveswere obtained by plotting the average percent relaxation (+/−SE) againstthe concentrations used.

The results of the ex vivo screening demonstrates that both A1 and A2dimers and A-type trimer induced vasorelaxation at a concentration of10⁻⁴M: at that concentration A1 dimer induced 93.20+/−3.46%vasorelaxation, A2 dimer induced 85.00+/−2.91% vasorelaxation, andA-type trimer induced 56.5+/−16.56% relaxation. Moreover, both A1 and A2dimers were more potent relaxants that the positive control(acetylcholine) and that difference was statistically significant atp=0.005 for A1 dimer.

The relaxation responses were attenuated when the vessels were eitherdenuded or pre-treated with L-NAME. Acetylcholine typically produced arelaxation response at a concentration of 10⁻⁷M, reaching maximalrelaxation at a concentration of 10⁻⁶M. No response was observed whenvessels were treated with vehicle alone.

Dose dependent relaxation mediated by the test compounds is representedin FIGS. 1A-C.

Example 4 Effects of A1 Dimer on Platelet in Whole Blood

Platelet aggregation of A1 dimer [A1D] was measured using a plateletcounting technique, and formation of platelet/monocyte conjugates (P/M)and platelet/neutrophil conjugates (PIN) by flow cytometry. In laterexperiments the activation state of platelets associated with leukocytes(CD62P) was also measured and also the activation state of theleukocytes themselves (CD11b).

Materials and Methods

A1 dimer [A1D] was dissolved in ethanol. Once in solution, furtherdilution with saline was possible. Hirudin, (Revasc™) was obtained fromNovartis (Basel, Switzerland) and was stored as a 5 mg/ml solution insaline in a glass vial at −20° C. Collagen (Nycomed) was from AxisShield Diagnostics (Dundee, UK). Concentrations were prepared from thestock solution (1 mg/ml) using the isotonic glucose buffer supplied bythe manufacturer. Aspirin (acetyl salicylic acid—ASA), adenosinediphosphate (ADP), platelet activating factor (PAF), arachidonic acid(AA) and epinephrine were from Sigma. Fixing solution consisted of 140mM NaCl containing 0.16% w/v formaldehyde, 4.6 mM Na₂EDTA, 4.5 mMNa₂HP04 and 1.6 mM KH₂PO4, pH 7.4.

Blood samples were studied using the Multi-Sample Agitator (MSA)produced by the Medical Engineering Unit (University of Nottingham). TheMSA is used to maintain blood samples at 37° C. and to agitate smallsamples of blood at a stir speed of 1,000 rpm as required. Flowcytometry was carried out using commercially available fluorescentlabelled antibodies on a Facscan (Becton Dickinson, UK) equipped with a5 W laser operating at 15 mW power and a wavelength of 488 nM or anLSRII flow cytometer (Becton Dickinson, UK) equipped with an additionalred Trigon laser operating at a wavelength of 633 nM.

Blood Collection

Blood was obtained from healthy volunteers, who denied taking anyaspirin or NSAIDS in the previous 10 days. This blood was dispensed intograduated polystyrene tubes that contained hirudin (final concentration50 μg/ml) and a small volume of the flavanol under investigation orethanol as control. The final concentration of ethanol in the blood wasalways 0.3%. In some experiments, aspirin (ASA) or saline as control wasalso included in the tube. The tubes were then capped and inverted threetimes to ensure adequate mixing then placed in the MSA at 37° C. for 30min before the experiments were performed, during which time the bloodwas left undisturbed. A further sample of blood was taken into acommercially prepared vacutainer tube that contained K2EDTA asanticoagulant.

Platelet Aggregation

Following the 30 min pre-incubation, aliquots of blood (480 μl) weredispensed into small polystyrene tubes each containing a stir bar andstirred for 2 min in the MSA. After 2 min a solution (20 μl) of agonistor vehicle control were added to the tubes. These were then stirred inthe MSA for up to 10 min at which time the platelet aggregates werefixed by mixing a small sub sample with fixative solution in a 1:2 ratio(v/v). The platelet count in the fixed samples was determined using theUltraFlo-100 Whole Blood Platelet Counter. Platelet aggregation wascalculated as the percentage loss of single platelets with reference tothe platelet count of the EDTA sample.

Platelet-Leukocyte Conjugate Formation

Platelet-leukocyte conjugate formation was measured in the same stirredsamples used to measure platelet aggregation. Sub samples were taken 4min or 10 min following the addition of agonist and transferred into theappropriate antibody or antibody mixture. These were then incubated inthe dark at room temperature for not less than 20 min. Following redcell lysis and a washing procedure the cell suspensions were applied toeither the FACScan or the LSRII flow cytometer. Leukocytes wereidentified by logical gating from dot plots of forward scatter (cellsize) and side scatter (cell granularity) profiles acquired with linearamplification. Monocytes were identified by their forward scatter-sidescatter profile and CD14 (PE) positivity, while neutrophils wereidentified in the same way but were negative for CD14 expression. The“pan” leukocyte marker, CD45 (PerCP) was also used to identify theleukocyte population. Fluorescence parameters were acquired withlogarithmic amplification. Platelet monocyte (P/M) and plateletneutrophil (P/N) conjugates were quantified as median CD42a (FITC)fluorescence of the monocyte (P/M mf) or neutrophil population (PIN mf).Leukocyte activation was measured by CD11b (AlexaFluor647) expression(CD11b-M for monocytes and CD11b-N for neutrophils). Platelet activation(P-selectin expression) was measured by CD62P (PE) positivity of theplatelets associated with leukocytes as (CD62P-M on P/M and CD62P-N onPIN).

The FACScan was used to measure the fluorescent probes in experimentswhere three colours were used together, but the LSRII was needed inorder to study four colours. The LSRII is a more sensitive machine andproduces higher fluorescence values (mf) than the FACScan. Resultsobtained on the FACScan cannot be directly compared with the resultsobtained on the LSRII.

Results Effects of A1 Dimer on Aggregation, P/M and PIN

Blood was obtained from three different volunteers and the plateletaggregation and platelet/leukocyte conjugate formation was measured inresponse to collagen (0, 0.125, 0.25 and 0.5 μg/ml). In theseexperiments aspirin was used at a concentration of 100 μM and A1 dimerwas used at 0.3 mM. Aggregation was measured at 4 and 10 min followingagonist addition and platelet/leukocyte conjugate formation only at 10min.

The absolute response of the blood from the different volunteers tocollagen varied. This meant that the relative inhibitory effects of aflavanol was dependent on the volunteer's responsiveness to theparticular collagen concentration used. For this reason it was decidedthat an appropriate means of analysing the results, for comparativepurposes, would be to calculate the mean values for A1 dimerirrespective of the collagen concentration used. The results are shownin FIG. 2. (Certain compounds other than A-type procyanidins were alsotested in these experiment, however, since they are not relevant to thediscussion here, they were not identified.) Because, for each of thethree blood samples three concentrations of collagen were used, theresults are each the means (±sem) of nine individual values.

A1 dimer inhibited collagen-induced platelet aggregation. ASA controlalso effectively inhibited the aggregation, P/M and PIN.

From this point on it was decided to include measures of the extent ofthe activation of both platelets and leukocytes in the conjugates thatformed following addition of collagen to blood. P-selectin (CD62P) wasmeasured on the platelets associated with leukocytes in the conjugatesthat formed. Leukocyte activation was measured as the amount of CD11bthat was expressed. Four-colour analysis was used.

Comparison of the Effects of Flavanols on Collagen-Induced Aggregation.P!M. PIN, CD62P-M, CD62P-N, CDIIb-M and CDIIb-N

Blood was obtained from three different volunteers and the plateletaggregation (4 min) and platelet/leukocyte conjugate formation (10 min)was measured in response to collagen (0, 0.125, 0.25 and 0.5 m/ml). Atthe same time the activation of platelets and leukocytes in theconjugates were measured by incubation with CD62P and CD11b antibodies.In these experiments aspirin was used at a concentration of 100 μM andA1 dimer was used at 0.3 mM. The results are shown in FIG. 3. (Certaincompounds other than A-type procyanidins were also tested in theseexperiment, however, since they are not relevant to the discussion here,they were not identified.) As before, the analysis was performed byincluding all results for all three collagen concentrations andcalculating the mean (±sem, n=9).

A1 dimer inhibited collagen induced aggregation. However, it wasnoteworthy that A1D did not inhibit leukocyte activation (CD11b onmonocytes and leukocytes). Aspirin also had no effect on CD11b.

1. A method of treating or preventing an NO-responsive disease ordisorder by administering to a subject in need thereof an effectiveamount of an A-type procyanidin composed of n monomeric units of theformula:

wherein (i) the monomeric units are connected via interflavan linkages4→6 and/or 4→8; (ii) at least two of the monomeric units areadditionally linked by an A-type interflavan linkage (4→8; 2→O→7) or(4→6; 2→O→7); (iii) n is 2 to 12; or a pharmaceutically acceptable saltor derivative thereof, and wherein the subject is a human or aveterinary animal.
 2. The method of claim 1, wherein the A-typeprocyanidin is isolated and purified.
 3. The method of claim 1, whereinthe A-type procyanidin is A1 dimer.
 4. The method of claim 3, whereinthe A1 dimer is isolated and purified.
 5. The method of claim 1, whereinthe A-type procyanidin is A2 dimer.
 6. The method of claim 5, whereinthe A2 dimer is isolated and purified.
 7. The method of claim 1, whereinthe A-type procyanidin is A-type trimer.
 8. The method of claim 7,wherein the A-type trimer is substantially pure.
 9. The method of claim7, wherein the A-type trimer has the formula:


10. The method of claim 1, wherein the NO-responsive disease or disorderis hypertension, cardiovascular disease, coronary artery disease,vascular circulation disorders and/or renal disease.
 11. The method ofclaim 1, wherein the subject is at risk of heart attack, stroke,congestive heart failure, and/or kidney failure.
 12. The method of claim1, wherein the subject suffers from high blood pressure in combinationwith any of the following: diabetes, obesity, high cholesterol levelsand/or smoking.
 13. A method of treating hypertension by administeringto a subject in need thereof an effective amount of an A-typeprocyanidin composed of n monomeric units of the formula:

wherein (i) the monomeric units are connected via interflavan linkages4→6 and/or 4→8; (ii) at least two of the monomeric units areadditionally linked by an A-type interflavan linkage (4→8; 2→O→7) or(4→6; 2→O→7); (iii) n is 2 to 12; or a pharmaceutically acceptable saltor derivative thereof, and wherein the subject is a human or aveterinary animal.
 14. The method of claim 1, wherein the A-typeprocyanidin is isolated and purified.
 15. The method of claim 1, whereinthe A-type procyanidin is A1 dimer.
 16. The method of claim 15, whereinthe AI dimer is isolated and purified.
 17. The method of claim 1,wherein the A-type procyanidin is A2 dimer.
 18. The method of claim 17,wherein the A2 dimer is isolated and purified.
 19. The method of claim1, wherein the A-type procyanidin is A-type trimer.
 20. The method ofclaim 19, wherein the A-type trimer is substantially pure.
 21. Themethod of claim 19, wherein the A-type trimer has the formula:


22. The method of claim 13, wherein the subject suffers from any of thefollowing: diabetes, obesity, high cholesterol levels and/or smoking.23. A pharmaceutical composition comprising an isolated and purified A1dimer.
 24. A pharmaceutical composition comprising an isolated andpurified A2 dimer.